CN113984728B - Construction method of fluorescent biosensor for rapid detection of listeria monocytogenes - Google Patents
Construction method of fluorescent biosensor for rapid detection of listeria monocytogenes Download PDFInfo
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- 241000186779 Listeria monocytogenes Species 0.000 title claims abstract description 34
- 238000001514 detection method Methods 0.000 title claims abstract description 30
- 238000010276 construction Methods 0.000 title abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims abstract description 12
- 108091023037 Aptamer Proteins 0.000 claims abstract description 10
- 239000002105 nanoparticle Substances 0.000 claims abstract description 5
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- 108010059993 Vancomycin Proteins 0.000 claims abstract description 4
- 229960003165 vancomycin Drugs 0.000 claims abstract description 4
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 claims abstract 2
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- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 claims description 4
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- General Health & Medical Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention relates to a construction method of a fluorescent biosensor for rapidly detecting listeria monocytogenes. By constructing an up-conversion nanoparticle (UCNPs) -mediated fluorescent biosensor, the fluorescent biosensor is used for rapid and sensitive detection of listeria monocytogenes. Preparing MNPs-Van magnetic nano probes by taking vancomycin (Van) as a first recognition molecule; biotinylated aptamer (aptamer) as a second recognition molecule can bind to internalizing protein a on the cell wall of listeria monocytogenes. The two recognition molecules are utilized to target different sites on target bacteria respectively, so that target bacteria are captured specifically, and a sandwich type complex (MNPs-Van/Listeria monocytogenes/aptamer) is formed. The HRP-TMB enzyme catalytic system is introduced to generate blue substances through the horseradish peroxidase marker (HRP-SA), and the fluorescence internal filtering effect of UCNPs is combined to reduce the fluorescence intensity of the UCNPs, so that the quantitative detection of the listeria monocytogenes is realized.
Description
Technical Field
The invention belongs to the technical field of food safety detection and analysis. In particular to a construction method of a fluorescent biosensor based on a double-site identification strategy and mediated by a fluorescent inner filtering effect, which is used for rapidly detecting listeria monocytogenes.
Background
The people take food as the day and take food as the first place, and the food safety is one of the most concerned civil problems of the common people. The food-borne disease caused by the food-borne pathogenic microorganisms is an important public health problem, and is also an important monitoring object for food safety in various countries. Food poisoning and food hygiene events are reported every year, especially pathogenic microorganisms are the main cause, and the method is extremely unfavorable for the orderly development of national health. From the data analysis of food-borne outbreaks in various countries, the causative factors of outbreaks are mainly microbial factors, whether in the united states, the european union, or china. Common food-borne pathogenic microorganisms include salmonella, escherichia coli, staphylococcus, vibrio parahaemolyticus, listeria monocytogenes, aspergillus flavus, viruses, avian influenza viruses, foot-and-mouth disease viruses, and the like. According to GB29921-2013, in general, except that staphylococcus aureus and vibrio parahaemolyticus can be detected within an allowable range, the fact that pathogenic bacteria cannot be detected in food is a very important food sanitation quality index. The food industry has long chain, multiple pollution links, rich nutrition of the food and suitability for growth of pathogenic bacteria, so that a plurality of different methods are needed for detecting the pathogenic bacteria in the food, which is very important for food safety.
Listeria monocytogenes, which is one of four world-wide food-borne pathogenic bacteria, is the only pathogen for human and livestock co-occurrence in Listeria, and can cause human beings to suffer from influenza-like diseases and serious complications, such as meningitis, septicemia, even natural abortion of pregnant women, and the like, and the mortality rate is as high as 30%. In addition, listeria monocytogenes is widely distributed, can survive and reproduce in various environments such as high salt, low temperature, acid and alkali, and is commonly called as a refrigerator killer; the commonly contaminated foods include meat products, dairy products and other whole industry chains, instant foods, frozen and refrigerated foods, so it is very important to establish a method for rapidly, sensitively and reliably detecting listeria monocytogenes.
At present, common detection methods of listeria monocytogenes mainly comprise: traditional microorganism culture method, immune detection method based on antigen-antibody specificity recognition and nucleic acid amplification detection method based on base complementary pairing principle, biosensor, etc. The microorganism culture method does not need special instruments and equipment, has high detection sensitivity and is always a gold standard for bacteria detection; however, the process is complicated, the period is long (4-7 days), the error leakage detection is easy to occur, and special experimenters are required to complete the detection process, so that the practical application of the method is limited, and the rapid detection of food production and field analysis cannot be satisfied. Immunoassays mainly include enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatographic test strip methods. ELISA has the advantages of relatively simple operation, high flux and the like; the colloidal gold immunochromatographic test strip has the advantages of simple operation, high reaction speed, suitability for on-site rapid detection and the like. However, the sensitivity of the two is general, the high specificity depends on the high-quality antibody, the preparation of the antibody is complex, the preservation condition is severe, the shelf life is short, the cost is high, and the further popularization and the use of the detection technology are not facilitated. The detection of pathogenic bacteria is improved to a molecular level by using a nucleic acid amplification technology, so that the effective detection of low-concentration target bacteria can be realized. Common nucleic acid amplification methods such as qPCR, mPCR, loop-mediated isothermal amplification methods and the like are complex in operation, require special personnel and special instruments, have high primer and probe design requirements, are difficult, and are easy to generate false positive results.
In recent years, researchers at home and abroad have been working to explore methods for detecting food-borne pathogenic bacteria suitable for on-site analysis, wherein biosensors are becoming a research hotspot. The biosensor has the advantages of high analysis speed, low cost, good portability and the like, is easy to realize on-site and instant detection, and has been widely applied to the fields of food safety, in-vitro diagnosis and the like. Among them, fluorescent biosensors are receiving more and more attention because of advantages of high sensitivity, simple operation, rapid response, and the like. Compared with the traditional organic fluorescent dye or quantum dot, the fluorescent biosensor constructed based on the fluorescence characteristics of the up-conversion nano particles (UCNPs) has certain advantages that the lanthanide metal doped UCNPs do not need ultraviolet and visible excitation light, but can up-convert 980nm near infrared light into visible light or near infrared light regions, the luminescence property of the fluorescent biosensor is little influenced by environmental change, and the fluorescent biosensor has the advantages of photon stability and thermal stability, non-flash and non-autofluorescence analysis, narrow emission spectrum, long fluorescence service life and the like, and can remarkably improve the signal to noise ratio of the sensor. Therefore, we design a fluorescent inner filtering effect based on UCNPs, converts the traditional ultraviolet absorbance signal into an up-conversion fluorescent signal, and finally establishes a relation with the concentration of target bacteria by utilizing the variation of the fluorescence intensity, thereby realizing the quantitative detection of the listeria monocytogenes, and achieving the purpose of rapidly and sensitively detecting the listeria monocytogenes in food by combining magnetic separation and non-immune recognition reaction.
Disclosure of Invention
The invention aims to provide a construction method of a fluorescent biosensor for rapidly detecting listeria monocytogenes and application thereof, which can realize rapid non-immune detection of the listeria monocytogenes. The sensor has a wider linear range, and has higher sensitivity and higher detection speed.
In order to achieve the above object, the present invention provides a technical solution comprising: the MNPs-Van nano probe is prepared by taking magnetic nano particles as a carrier (MNPs), and a specific aptamer (aptamer) is combined, so that the double-site non-immune recognition of the listeria monocytogenes is realized. The fluorescent internal filtering effect of UCNPs, namely that the emission wavelength of UCNPs overlaps with the absorption wavelength of colored products, induces the change of fluorescence intensity, converts the traditional ultraviolet absorbance signal into an up-conversion fluorescence signal, and establishes a standard curve for quantitative detection of listeria monocytogenes.
For detection of listeria monocytogenes, the invention has the advantages compared with the prior art;
1. the invention uses vancomycin and nucleic acid aptamer as biological recognition molecules, avoids the use of antibodies in traditional immunoassay, and reduces the cost.
2. The invention combines a double-site identification strategy with the fluorescence internal filtering effect of UCNPs, realizes signal conversion and signal amplification, overcomes the defects of low sensitivity, narrow linear range and the like of the traditional colorimetric sensing method, and can meet the detection of Listeria monocytogenes in a certain concentration range.
3. The fluorescent biosensor established by the invention is convenient to manufacture, small in required sample quantity, strong in external interference resistance and easy to realize on-site instant detection of food-borne pathogenic bacteria.
Drawings
FIG. 1 is a schematic diagram of the principle of the fluorescent biosensor prepared by the present invention for detecting Listeria monocytogenes.
FIG. 2 is a representation of UCNPs: (a) TEM images of OA-UCNPs; (B) X-ray diffraction results of UCNPs; (C) TEM images of ADA-UCNPs; (D) fourier transform infrared spectra of UCNPs; (E) fluorescence spectrum of UCNPs; (F) ADA-UCNPs, MNPs and MNPs-Van's Zeta potential.
FIG. 3 is a representation of MNPs and MNPs-Van: (a) and (B) TEM images of MNPs and MNPs-Van; (C) hydrated particle sizes of MNPs and MNPs-Van; (D) fourier transform infrared spectra of MNPs and MNPs-Van; (E) electron microscopy images of listeria monocytogenes; (F) MNPs-Van conjugates capture electron microscopy images of Listeria monocytogenes.
Fig. 4 is an optimized experimental parameter: (a) concentration of MNPs-Van conjugates; concentration of aptamer (B) concentration of HRP-SA.
FIG. 5 is a sensitivity analysis of a constructed fluorescence sensor to detect Listeria monocytogenes: (A) amount of change in fluorescence intensity; (B) a standard curve.
FIG. 6 is a graph showing the results of the specific detection of Listeria monocytogenes by the constructed fluorescence sensor.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It should be apparent to those skilled in the art that the examples are merely provided to aid in understanding the present invention and should not be construed as limiting the invention in any way.
Example 1
1. Preparation of fluorescent biosensor
(1) Preparation of UCNPs
1) 0.1164g YCl were weighed out respectively 3 ·6H 2 O、0.0892g GdCl 3 ·6H 2 O、0.062g YbCl 3 ·6H 2 O and 0.006g ErCl 3 ·6H 2 O is dissolved in 4mL of methanol solution, ultrasonic wave is carried out until the O is completely dissolved, and then the O is poured into a three-neck flask which is rinsed by a little methanol (the three-neck flask must be kept clean and can be soaked with aqua regia overnight if necessary); then pouring 6mL of oleic acid and 14mL of 1-octadecene to obtain a mixture A;
2) Under continuous magnetic stirring, the mixture A is heated to 160 ℃ under the protection of nitrogen, kept for 30min and then cooled to about 50 ℃;
3) 0.2964g NH are weighed respectively 4 F and 0.2g NaOH are dissolved in 20mL methanol solution, and the solution is sonicated for about 15min to complete dissolution; the solution was then added dropwise to the flask solution described above with continuous stirring. Then the mixture is placed under the water bath condition of 50 ℃ for 40min, and then is kept under the water bath condition of 70 ℃ for 60min so as to facilitate the volatilization of methanol, thus obtaining a mixture B;
4) Subsequently, mixture B was heated to 300 ℃ and held for 60min with continuous stirring. Cooling to room temperature, centrifuging at 10000rmp for 3min, and removing supernatant; and the precipitate was washed 3 times with cyclohexane and ethanol (10000 rmp centrifuged for 3 min), and finally the collected solid was dried overnight under vacuum at 60 ℃ and stored under sealed conditions.
The above synthesized up-conversion nanoparticles are oleic acid-coated (OA-UCNPs) and therefore hydrophobic, which is detrimental to subsequent further use. In order to convert hydrophobic UCNPs into hydrophilic UCNPs, the present study surface-modified UCNPs by ligand exchange, while alendronate (ADA) was used as a ligand to displace the original hydrophobic ligand on the surface of UCNPs. The specific method comprises the following steps: 50mg of ADA and 200mg of OA-UCNPs solid are weighed, mixed and dispersed in 10mL of chloroform, 4mL of ethanol and 6mL of ultrapure water, and the mixture is subjected to ultrasonic treatment for about 5min; the pH was adjusted to 2-3 with 1M HCl and reacted for 30min with continuous stirring. After the reaction was completed, the mixture was washed with ethanol and pure water 3 to 4 times (10000 rmp centrifugation for 3 min). Finally, the obtained product (ADA-UCNPs) was redispersed in 10mL of ultra-pure water and placed at 4℃for further use.
(2) Preparation of MNPs-Van conjugates
First, 1mg of carboxyl-coated Magnetic Nanoparticle (MNPs) suspension was transferred to a 1.5mL centrifuge tube, washed 2 times with MEST (10 mm mes, ph=6.0, 0.05% tween-20) and transferred to a new 1.5mL centrifuge tube. After magnetic separation, carboxylated magnetic nanoparticles were activated with 50. Mu.L EDC and NHS (10 mg/mL) for 20min at room temperature. The test tube was placed on a magnetic separation rack, washed 2 times with MEST, then dispersed in phosphate buffer (10 mm pbs, ph=7.4) containing 1.0mg vancomycin, and incubated for 6h with continuous gentle shaking at room temperature. After magnetic separation and removal of the supernatant, 1mL PBST and 1% bsa were added to the tube and incubated for 30min to block the residual sites. Finally, MNPs-Van conjugates were washed 3 times with PBST, then dispersed in 1mL PBST and 0.5% BSA, and stored at 4℃for use.
2. Sample detection
(1) Detection method
1) Gradient dilution of Listeria monocytogenes bacterial fluid of known concentration to 10 2 ，2×10 2 ，2×10 3 ，2×10 4 ，2×10 5 ，2×10 6 ，2×10 7 And 2X 10 8 CFU/mL。
2) mu.L of MNPs-Van conjugate was conjugated to 400. Mu.L of each conjugate (10 2 -2×10 8 CFU/mL) of the target bacterial liquid was mixed in a 1.5mL sterile centrifuge tube and incubated at 37 ℃ for 30min with continuous shaking.
3) After magnetic separation, the mixture was washed 3 times with PBST buffer, 100mL of biotinylated aptamer was added, and the mixture was shaken and incubated at 37℃for 30min.
4) Magnetic separation, washing 3 times, adding 100. Mu.L HRP-SA solution, shaking at 37℃and incubating for 30min. The complex was magnetically separated, washed 3 times, redispersed with 100. Mu.L deionized water and reacted with 200. Mu.L TMB solution in the dark for 10min.
5) After magnetic separation, 200. Mu.L of the solution was aspirated from the supernatant and mixed with 100. Mu.L of UCNPs solution (1 mg/mL) to give mixture C. The up-converted fluorescence emission spectrum of mixture C was then measured on a fluorescence spectrophotometer with a 980nm laser source.
Each spot was analyzed 3 times (n=3), and for each interval, the fluorescence intensity variation (Δfl intensity) was calculated as: Δfl intensity= |fl intensity sample -FL intensity blank |。
(2) Condition optimization
On the basis of the detection method (1), the concentration of the MNPs-Van conjugate, the concentration of the aptamer and the dilution ratio of HRP-SA are optimized, and as shown in figure 4, the concentrations of the MNPs-Van conjugate and the aptamer are respectively 0.1mg/mL and 0.1mM, and the dilution ratio of HRP-SA is 1:2000.
(3) Standard Curve establishment
Based on the detection method (1), a standard curve between the amount of change in fluorescence intensity and the concentration of Listeria monocytogenes is established by plotting the logarithm of the concentration of the sample (CFU/mL) as the abscissa and the value of ΔFL intensity as the ordinate, as shown in FIG. 5.
(4) Specificity verification
The specificity of the method was verified by using gram positive bacteria (staphylococcus aureus and bacillus stearothermophilus) and gram negative bacteria (escherichia coli and salmonella enterica) as negative control groups. As shown in FIG. 6, ΔFL intensity when detecting Listeria monocytogenes is significantly higher than the negative control.
(5) Actual sample detection
The recovery rate was studied using standard addition methods, i.e., different concentrations of listeria monocytogenes were added to the blank ham samples. As shown in the following table, the average recovery rate of detecting Listeria monocytogenes in the ham sample is 88.0% -108.5%, which shows that the method has certain feasibility and accuracy.
The applicant states that the process of the invention is illustrated by the above examples, but the invention is not limited to, i.e. does not mean that the invention must be carried out in dependence on the above process steps. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.
Claims (1)
1. A method for constructing a fluorescent biosensor for rapidly detecting listeria monocytogenes is characterized by comprising the following steps:
step one, preparation of a sensor
(1) Preparation of UCNPs
The lanthanide metal doped up-conversion nano-particles UCNPs are synthesized by a thermal decomposition method, and the specific preparation method is as follows:
1) 0.1164g YCl were weighed out respectively 3 ·6H 2 O、0.0892g GdCl 3 ·6H 2 O、0.062g YbCl 3 ·6H 2 O and 0.006g ErCl 3 ·6H 2 Dissolving O in 4mL of methanol solution, performing ultrasonic treatment until the O is completely dissolved, pouring the solution into a three-neck flask which is rinsed with a little methanol, and pouring 6mL of oleic acid and 14mL of 1-octadecene to obtain a mixture A;
2) Under continuous magnetic stirring, placing the mixture A under the protection of nitrogen, heating to 160 ℃, keeping for 30min, and then cooling to about 50 ℃;
3) 0.2964g NH are weighed respectively 4 F and 0.2g of NaOH are dissolved in 20mL of methanol solution, and the solution is dissolved completely by ultrasonic treatment for about 15min, then the solution is added into the flask solution dropwise under continuous stirring, and then the mixture is kept for 40min under the water bath condition of 50 ℃ and then is kept for 60min under the water bath condition of 70 ℃ so as to facilitate the volatilization of methanol, thus obtaining a mixture B;
4) Subsequently, heating the mixture B to 300 ℃ and keeping the mixture B for 60min under continuous stirring, cooling the mixture B to room temperature, centrifuging 10000rmp for 3min, removing supernatant after centrifugation, washing precipitate with cyclohexane and ethanol for 3 times, and finally drying the collected solid at 60 ℃ overnight in vacuum, and sealing and preserving the solid;
the synthesized up-conversion nano particles are OA-UCNPs wrapped by oleic acid, and the UCNPs are subjected to surface modification by adopting a ligand exchange mode, namely 50mg of ADA and 200mg of OA-UCNPs are weighed and mixed, and dispersed in 10mL of chloroform, 4mL of ethanol and 6mL of ultrapure water, and are subjected to ultrasonic treatment for about 5min; adjusting the pH to 2-3 by using 1M HCl, and reacting for 30min under continuous stirring; after the reaction is finished, washing with ethanol and pure water for 3-4 times, centrifuging for 3min at 10000rmp, finally, re-dispersing the obtained ADA-UCNPs product in 10mL of ultrapure water, and placing at 4 ℃ for standby;
(2) Preparation of MNPs-Van conjugates
Firstly, transferring 1mg of carboxyl-coated magnetic nanoparticle MNPs suspension into a 1.5mL centrifuge tube, washing 2 times with 10mM MES (medium density polyethylene) prepared by 0.05% Tween-20 (medium density polyethylene) and transferring into a new 1.5mL centrifuge tube, magnetically separating, activating carboxylated magnetic nanoparticles with 50 mu L EDC and 10mg/mL NHS for 20min at room temperature, placing the tube on a magnetic separation rack for separation, washing 2 times with MEST, dispersing in phosphate buffer solution containing 10mM PBS and pH=7.4 containing 1.0mg vancomycin, continuously and gently oscillating and incubating for 6h at room temperature; after magnetic separation and removal of the supernatant, 1mL of PBST and 1% bsa were added to the test tube, incubated for 30min, the residual sites were blocked, MNPs-Van conjugates were washed 3 times with PBST, then dispersed in 1mL of PBST and 0.5% bsa, and stored at 4 ℃ for later use;
step two, detection method of listeria monocytogenes
First, a known concentration of Listeria monocytogenes bacteria solution is diluted to 10 in a gradient 2 ，2×10 2 ，2×10 3 ，2×10 4 ，2×10 5 ，2×10 6 ，2×10 7 And 2X 10 8 CFU/mL; then 100. Mu.L of MNPs-Van conjugate was combined with 400. Mu.L of 10, respectively 2 -2×10 8 Mixing target bacterial solutions with different concentrations of CFU/mL in a 1.5mL sterile centrifuge tube, and continuously oscillating and incubating for 30min at 37 ℃; washing with PBST buffer solution for 3 times after magnetic separation, adding 100mL of biotinylated aptamer, shaking at 37deg.C, and incubatingCulturing for 30min; magnetic separation and washing for 3 times, adding 100 mu L of HRP-SA solution, oscillating at 37 ℃ and incubating for 30min; magnetically separating, washing for 3 times, redispersing the compound with 100 mu L of deionized water, and reacting with 200 mu L of TMB solution for 10min under dark condition; after magnetic separation, 200. Mu.L of the solution is sucked from the supernatant and is uniformly mixed with 100. Mu.L of 1mg/mL UCNPs solution to obtain a mixture C; subsequently, the mixture C is placed on a fluorescence spectrophotometer with a 980nm laser source, and the up-conversion fluorescence emission spectrum of the mixture C is measured; each spot was analyzed 3 times (n=3), and for each interval, the fluorescence intensity variation (Δfl intensity) was calculated as: Δfl intensity= |fl intensity sample -FL intensity blank |；
Step three, data monitoring and recording
The fluorescence intensity (FL intensity) is measured by an autonomously built fluorescence detection system.
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